Serum HSP90-Alpha and Oral Squamous Cell Carcinoma : A Prospective Biomarker

Aim: Current study aims to perform differential protein expression analysis of serum samples from Oral squamous cell carcinoma (OSCC) patients and healthy controls in search of potential diagnostic and/or prognostic biomarker(s). Objective: OSCC is diagnosed late, resulting in poor survival and high mortality. Identification of non-invasive prognostic biomarker is of utmost importance for early diagnosis and proper management of the disease; hence we used proteomic approach to identify potential biomarkers from serum. Methods: Serum samples (OSCC n=45 and control n=30) were depleted and proteins were separated using 2-D gel electrophoresis followed by identification by mass spectrometric analysis. Gene expression analysis of identified proteins in malignant and normal tissue was also performed to complement proteomics studies. Results: Among differentially expressed proteins, a noteworthy observation was up regulation of Heat shock protein alpha (HSP90α) from serum of oral cancer patients. We also observed elevated levels of Haptoglobin (HP) along with down regulation of Type II keratin cytoskeletal 1(KRT1) and serum Albumin (ALB) in oral cancer patients. Gene expression studies of identified proteins in malignant and normal tissue revealed a similar pattern with the exception of KRT1. We believe that elevated levels of serum HSP90 alpha might be used as a potential biomarker. Conclusion: Our findings suggest the contribution of HSP90 alpha and other identified proteins in oral pathology as pro/anti apoptotic modulators, thus they are being considered as predictive biomarkers.

Severe Hyperthermia Induces Apoptosis Mediated by Caspases Activation and Suppression of Hsp90-alpha Expression in Osteosarcoma Cells

BACKGROUND: Hyperthermia is used as an adjuvant treatment to sensitize cancer cells to subsequent radiotheraphy or chemotherapy. The aim of this study was to study the effect of severe hyperthermia on osteosarcoma cells and its underlying causes.METHODS: Short-term (1 h) severe hyperthermia (45°C) was applied to osteoblast-like osteosarcoma cells (MG-63) and the heat shock response and cell death mechanisms were investigated after recovery at 37°C for 72 h.RESULTS: Cell viability was significantly reduced (p<0.05) and apoptosis was significantly induced by severe hyperthermia in MG-63 cells (p<0.001). Caspase 3/7, 4 and 12 activities were significantly increased after 72 h of recovery at 37°C, indicating that severe hyperthermia induced endoplasmic reticulum (ER) stress and apoptosis (p<0.05). Heat shock protein 90 alpha (Hsp90α) was significantly down regulated at the protein level after recovery, in association with apoptosis induction (p<0.01). Additionally, caspase 8 and 9 were activated, possibly as a result of ER stress or other stimuli while, B-cell leukemia 2 family protein (BCL-2) mRNA was down regulated (p<0.01).CONCLUSION: Severe hyperthermia could cause prolonged cell cytotoxicity by inducing apoptosis in association with inhibition of Hsp90α. This indicates the therapeutic potential of severe hyperthermia for the treatment of osteosarcoma.KEYWORDS: hyperthermia, apoptosis, endoplasmic reticulum, stress, heat shock proteins, osteosarcoma

HSP90 Chaperone – An Important Player in CML: Study At RNA and Protein Level

Abstract 4878 Chronic myeloid leukemia (CML) is the myeloproliferative disease characterised by presence of BCR-ABL tyrosine kinase, which has been proved to play the basic role in CML ontogenesis. BCR-ABL as well as other kinases active during disease progression (e.g. Src kinase) belong to HSP90 client proteins. It is known, that HSP90 protein is overexpressed in cancer cells and leukemias and is related to therapy resistance in CML cells (Gorre et.al. Blood 2002;100:3041-3044). HSP90 exists in two isoforms: alpha - inducible and beta – constitutively expressed. Taherian et.al. (Biochemistry and Cell Biology, 2008, 86:(1) 37–45) demonstrated that Hsp90α and Hsp90β exhibit similar interactions with cochaperones, but significantly different substrate specificity under stress conditions. Those results reveal both functional similarities and key functional differences between the individual members of this protein family. In our previous study we found high protein expression of total HSP90 in leukocytes of CML patients in advanced disease states and in patients with poor responses (Zackova et.al. EHA 2011). The HSP90 seems to be an indicator of disease deterioration in patients with chronic myeloid leukemia. In the current study, we extended our field of interest on HSP90 isoforms alpha and beta on mRNA level. We aimed to find out possible correlation between protein and mRNA expression levels, as well as between mRNA levels and response to the therapy. We wondered to know whether the monitoring of total HSP90 or its isoforms in CML can early predict the disease deterioration. We tested HSP90 alpha and HSP90 beta mRNA expression levels in patients with various response of CML by real-time RT-PCR method. The mRNA profiles showed high similarity with the data obtained from previous western blot analyses. The analyses showed that high HSP90 levels are associated with poor response to therapy and in advanced disease phases. These levels are probably represented by HSP90-beta isoform (constitutively expressed), which is expressed in higher levels comparing to HSP90 alpha in all samples tested. Expression of HSP90 alpha isoform is much lower while its mRNA expression level highly increases only in blast crisis. Studying both isoformes separately could distinguish various mechanisms in disease progression. The results of this and previous studies suggest HSP90 (on protein and mRNA levels) is an important molecule for studying of prognosis in CML patients. Thus HSP90 appears to be a candidate for novel marker of CML progression. Disclosures: No relevant conflicts of interest to declare.